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Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K#5 and K#14. In keratinocyte autocrine culture, the exogenously added epidermal growth factor (EGF) has no effect on cell proliferation and mitogen-activated protein kinase (MAPK) activity. PD98059 inhibits MAPK pathway and autocrine keratinocyte proliferation. Staurosporine and Gö6976 strongly inhibit autonomous keratinocyte proliferation. In contrast, Gö6983 (which does not inhibit PKC micro ) inhibits only 20% of autocrine keratinocyte proliferation. Staurosporine inhibits MAPK activity, whereas Gö6976 and Gö6983 strongly increase it. We have concluded that MAPK, PKC micro and probably PKCalpha take part in autocrine keratinocyte proliferation. The effect of Gö6976 and Gö6983 on MAPK activity could be explained by the inhibition of PKC-dependent MAPK-phosphatase expression. The effect of staurosporine could be explained by its paradoxical action (activation) on protein kinase C (PKC) in keratinocytes.

Autocrine growth of human epidermal keratinocytes can be maintained in subconfluent cell cultures in the absence of exogenous growth factors. We used this culture model to investigate the interactions between the mitogen-activated protein kinase (MAPK) pathway and Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) in autocrine keratinocyte proliferation. We have previously demonstrated that MAPK and protein kinase C (PKC) are both involved in keratinocyte proliferation in a complex set of interactions. Treatment of keratinocytes with PD98059, a potent inhibitor of MAPK kinase, inhibited the MAPK pathway, c-myc activation and autocrine keratinocyte proliferation. Application of the CaM-kinase inhibitor KN-62 also led to a strong inhibition of MAPK/c-myc activation and autocrine keratinocyte proliferation. Other inhibitors, such as wortmannin (selective and potent inhibitor of phosphatidylinositol 3-kinase) and AG 490 (JAK2 inhibitor) had weak effects on autocrine keratinocyte proliferation, MAPK and c-myc activation. Our results clearly demonstrate a crosstalk between CaM-kinase/MAPK pathways in transducing keratinocyte proliferation stimuli.

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